Internalization of the thrombopoietin receptor is regulated by 2 cytoplasmic motifs

Receptor-mediated internalization appears to be the primary mean of regulating the plasma level of thrombopoietin (TPO). However, the processes that regulate MpI internalization have not previously been described. Using the cytokine-dependent cell line BaF3, we have identified 2 distinct motifs within the cytoplasmic domain of MpI that underlie ligand-dependent internalization. Removal of the fourth cytoplasmic tyrosine residue by deletion or truncation results in a significant decrease in maximal internalization. The remaining receptor internalization is abrogated by deletion of cytoplasmic residues 54-69, which include the core box2 region ((LLEIL58)-L-54-E-55-I-56-L-57) and the only dileucine motifs ((LL55)-L-54 and (IL58)-L-57) within the cytoplasmic domain of MpI. Receptor internalization mediated by this latter subdomain does not require Jak2 activation. Furthermore, TPO-stimulated cellular proliferation appears to be directly correlated with receptor internalization, indicating that internalization of the TPO/MpI complex may be essential for normal signal transduction. Finally, we have demonstrated that upon removal of TPO from the supernatant, MpI promptly reappears on the cell surface, suggesting that a pool of intracellular MpI can be rapidly recycled to the cell surface. These data help identify the receptor motifs involved in TPO-induced internalization of MpI and suggest that MpI translocation may be necessary for normal cellular proliferation. (C) 2003 by The American Society of Hematology.